Lentiviral packaging constructs

ABSTRACT

The present invention provides novel lentiviral packaging constructs that are useful for the establishment of stable packaging cell lines and producer cell lines. In particular, the present invention provides novel packaging cell lines that are capable of constitutively expressing high levels of lentiviral proteins.

This application claims the benefit under 35 USC §119(e) of U.S.provisional patent application No. 60/275,275, filed Mar. 13, 2001, for“Lentiviral Packaging Constructs.” The disclosure of this provisionalapplication is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to novel lentiviral packaging constructs, stablepackaging cell lines, stable producer cell lines and the use thereof forproducing recombinant lentiviral vectors in mammalian cells.

BACKGROUND OF THE INVENTION

Lentiviruses are complex retroviruses which, in addition to the commonretroviral genes gag, pol and env, contain other genes with regulatoryor structural function. The higher complexity enables the lentivirus tomodulate the life cycle in the course of latent infection. A typical andwell-characterized lentivirus is the human immunodeficiency virus (HIV),however, several animal lentiviruses have been described as well.

Viral vectors derived from lentiviruses are a useful tool for genedelivery. The ability of lentiviral vectors to deliver a gene into abroad range of rodent, primate and human somatic cells makes thesevectors well suited for transferring genes to a cell for gene therapypurposes. Lentiviruses can infect terminally differentiated cells thatrarely divide, such as neurons and macrophages, which renders themparticularly useful for certain gene therapy applications requiring thetransduction of non-dividing cells.

For producing recombinant lentiviral vectors packaging cell lines areused which supply in trans the proteins necessary for producinginfectious virions. An important consideration in the construction ofretroviral packaging cell lines is the production of high titer vectorsupernatants free of recombinant replication competent retrovirus (RCR).One approach to minimize the likelihood of generating RCR in packagingcells is to divide the packaging functions into at least two constructs,for example, one which expresses the gag and pol gene products and theother which expresses the env gene product. This approach minimizes theability for co-packaging and subsequent transfer of the two genomes, aswell as significantly decreasing the frequency of recombination betweenthe viral genomes in the packaging cell to produce RCR. In the eventrecombinants arise, mutations or deletions can be configured within theundesired gene products to render any possible recombinantsnon-functional. In addition, deletion of the 3′ LTR on the packagingconstructs further reduces the ability to form functional recombinants.

One of the major hurdles encountered in the art when producing a stablelentiviral-based packaging cell line is the inability to maintain highlevels of expression of Gag/Pol proteins. This could be due to theinherent toxicity of some of the lentiviral proteins or to diminishedprotein expression from promoter silencing. Accordingly, packagingsystems currently known in the art are either transient packagingsystems or employ inducible promoters to minimize toxicity problems(Naldini et al., Science 272:263-267, 1996; Kafri et al., Journal ofVirology 73:576-584, 1999). These approaches, however, aredisadvantageous because they require considerable effort and time forlentiviral vector production. Furthermore, vector batches obtained fromsuch systems will display a higher variability as compared to batchesthat would be obtainable from stable packaging cell lines. Furthermore,it is difficult to scale up lentiviral vector production from atransient system.

SUMMARY OF THE INVENTION

The present invention provides novel lentiviral packaging constructsthat are useful for the establishment of stable packaging cell lines andproducer cell lines. In particular, the present invention provides novelpackaging cell lines that are capable of constitutively expressing highlevels of lentiviral proteins, such as for example HIV p24 gag proteinin the case of a HIV based packaging cell line, or of BIV RT protein inthe case of a BIV based packaging cell line.

In one aspect the present invention provides a lentiviral packagingconstruct comprising a deletion in the lentiviral packaging signal and aportion of the lentiviral pol gene which includes the protease encodingsequence, wherein said protease encoding sequence includes a mutationcorresponding to a T26S substitution in the encoded lentiviral protease.In another aspect a stable pre-packaging cell line is providedcomprising the packaging construct of the invention.

In a further aspect, a stable packaging cell line comprising thepackaging construct of the invention and further comprising a plasmidcomprising an env gene is provided, as well as a producer cell linewhich additionally comprises a lentiviral plasmid vector.

In yet another aspect a lentiviral vector particle obtained from thestable producer cell line of the invention is provided.

Also provided is a method for producing a lentiviral vector particlepreparation comprising the steps of transfecting the stable packagingcell line of the invention with a lentiviral plasmid vector, propagatingthe cell line obtained thereby in a suitable culture medium andobtaining a lentiviral vector particle preparation from the said culturemedium.

DESCRIPTION OF THE FIGURES

FIG. 1 shows HIV-based vectors of the invention in a schematic view:FIG. 1A shows a series of packaging constructs: PHIVΔΨ; pΔVΔR furtherhaving a deletion of vif and vp; pΔVΔR-PR* further having a pointmutation in the active site of protease; pΔVΔR-SAR and pΔVΔR-PR*SARfurther including the interferon β SAR element. FIG. 1B shows thetransfer vector pHLEIP. FIG. 1C shows envelope constructs useful forpseudotyping.

FIG. 2 is a graph comparing the viral production, as measured by HIV p24levels, from the different HIV packaging cell lines over time(approximately 12 weeks).

FIG. 3 shows schematic of pCligpSyn.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology,cell culture, virology, and the like which are in the skill of one inthe art. These techniques are fully disclosed in current literature andreference in made specifically to Sambrook, Fritsch and Maniatis eds.,“Molecular Cloning, A Laboratory Manual”, 2nd Ed., Cold Spring HarborLaboratory Press (1989); Celis J. E. “Cell Biology, A LaboratoryHandbook” Academic Press, Inc. (1994) and Bahnson et al., J. of Virol.Methods, 54:131-143 (1995).

All publications and patent applications cited in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains and are hereby incorporated by reference intheir entirety.

The present invention is concerned with novel lentivirus-based packagingconstructs that are useful for the establishment of stable packagingcell line and producer cell lines. Surprisingly it is found thatmutations in the active site of the respective lentiviral protease geneenable the construction of lentiviral packaging vectors which are usefulto establish stable packaging cell lines for the production oflentiviral vectors.

The catalytic center of HIV protease includes a three amino acid motif,Asp-Thr-Gly (Konvalinka, J. et al.,. J. Virol. 69:7180-7186,1995) Thesethree amino acids are conserved among HIV and SIV isolates documented sofar (Korber B, Theiler J, Wolinsky S Science 1998 Jun. 19 280: 53711868-71). Konvalinka, J. et al. mutated the Thr residue (correspondingto amino acid number 26 from the start of Protease in HIV isolate HXB2)to a Ser. They found that the mutated HIV protease has a significantlyreduced toxicity while preserving the protease activity.

It has been surprisingly found that this information makes it possiblefor one to generate a stable cell line to express high levels oflentiviral Gag/Pol proteins. Expression of these proteins is absolutelynecessary in order to establish a stable packaging cell line forlentiviral vectors, in particular for HIV- or BIV-based lentiviralvectors.

Furthermore, surprisingly, it was found in the present invention thatthe Asp-Thr-Gly motif is also present in BIV protease in the samelocation. A comparison of the first 29 Amino Acids of HIV and BIVproteases reveals that the amino acids number 25 to 29 are identicalbetween HIV and BIV proteases, including the said Asp-Thr-Gly motif: HIVProtease (HXB2): 1-PQVTLWQRPLVTIKIGGQLKEALLDTGAD (SEQ ID NO:1) BIVProtease (127 isolate): 1-SYIRLDKQPFIKVFIGGRWVKGLVDTGAD (SEQ ID NO:2)HIV Protease mut 1-PQVTLWQRPLVTIKIGGQLKEALLDSGAD (SEQ ID NO:3) BIVProtease mut 1-SYIRLDKQPFIKVFIGGRWVKGLVDSGAD (SEQ ID NO:4)

Accordingly in one embodiment this invention provides for a mutation ofthe Thr to Ser in the BIV isolate 127 protease at the amino acid number26 from the start of protease (SEQ ID NO:4) to generate a less toxic BIVprotease as compared to wild type BIV protease. A BIV based stablepackaging cell line, for BIV based lentiviral vector production,expressing BIV Gag/Pol with this point mutant in the protease codingregion may then be generated. Such a stable packaging cell line allowsfor the development of a BIV lentiviral vector producing cell line.

In a further embodiment of the invention, it is found that combining theinclusion of protease genes having mutations in their active site withthe inclusion of SAR elements into the lentiviral packaging constructmay provide particularly advantageous results. Such packaging cell linesare capable of constitutively expressing particularly high levels oflentiviral proteins, such as for example the HIV p24 Gag protein. A highlevel of Gag (>5 ng/ml p24) is required for a stable packaging cell lineto produce efficient titers. Preferably, the stable packaging cell lineproduces >100 ng/ml p24 and more preferably >1 μg/ml p24.

In one embodiment, the present invention provides a series of HIV-basedpackaging constructs. These packaging constructs are transfected intosuitable cell lines (FIG. 1A). The original construct, pHIVΔΨ has beenextensively used for transient production of vector supernatant, whichhas been very efficient at transducing a variety of target cells andtissues. The first modification introduced in order to make thepackaging construct more suitable for stable vector production is thedeletion of two accessory proteins, vif and vpr, to make pΔVΔR. Neitherof these proteins is necessary for vector production (Zufferey et al,Nature Biotechnology. 15:871-875, 1997) and vpr has been shown to becytostatic and might prevent the production of a stable producer cellline (Rogel, M. E. et al, J. Virol. 69:882-888, 1995). To further limitthe potential toxicity of the construct, a point mutation is introducedinto the active site of protease to produce pΔVΔR-PR*. This mutation hasbeen reported to reduce the cytotoxicity caused by protease, but stillallow normal viral processing functions (Konvalinka, J. et al.,. J.Virol. 69:7180-7186, 1995).

In a particular embodiment of this invention, a further modification toimprove the stable expression of HIV Gag/Pol proteins is theintroduction of the interferon β SAR element (Klehr, D et al.,.Biochemistry. 30:1264-1270, 1991). For example, such a modificationresults in the two vectors, pΔVΔR-SAR and pΔVΔR-PR*SAR.

The packaging constructs are tested for their ability to package an EGFPexpressing vector and transduce 293T cells in the transient assay asdescribed in the Examples below.

All of the vector supernatants that have been generated with the use ofthese constructs exhibit transduction efficiencies greater than 90% asmeasured by FACS analysis for EGFP expression indicating that theabove-described modifications do not impair the normal packagingfunctions. Accordingly, it is found that stable packaging cell lines canbe obtained if the packaging construct contains an active site mutationin the protease, which prevents toxicity and a SAR element. The SARelement may serve to reduce promoter silencing, although Applicants donot wish to be bound by any theoretical speculation as to themechanistic explanation of the invention described.

Accordingly, in one aspect the present invention provides a lentiviralpackaging construct comprising a deletion in the lentiviral packagingsignal and a portion of the lentiviral pol gene which includes theprotease encoding sequence, wherein said protease encoding sequenceincludes a mutation corresponding to a T26S substitution in the encodedlentiviral protease.

A lentiviral “packaging construct”, also sometimes referred to as ahelper construct, refers to an assembly which is capable of directingexpression of one or more lentiviral nucleotide sequences that providein trans the proteins required to obtain lentiviral vector particles. Inone embodiment of the invention the nucleotide sequences include atleast the gag gene and/or pol gene of a lentivirus; a promoter operablylinked to the respective nucleotide sequences and generally apolyadenylation sequence located downstream of the respective nucleotidesequences encoding the gag and/or pol genes. The polyadenylationsequence, for example, may be derived from Simian virus 40 (SV40).

A mutation “corresponding to” a T26S substitution in the encodedlentiviral protease may be either the T26S substitution itself, which isthe preferred substitution of the invention, or a substitution having anequivalent biologic effect. “Equivalent biologic effect” means asubstitution resulting in a similar loss of protease cytotoxicity as theT26S substitution itself, while retaining a similar level of viralprotease activity as the T26S substitution itself. Cytotoxicity may bemeasured as described in Konvalinka, J. et al.,. J. Virol. 69:7180-7186,1995, in particular vimentin cleavage may be used as a marker forcytotoxicity. “Viral protease activity” may be measured as described inKonvalinka, J. et al.,. J. Virol. 69:7180-7186, 1995. In particular,cleavage of particle-associated polyproteins in the virus having themutation to be assessed is a suitable measure for viral proteaseactivity. Activities and cytotoxicities are “similar” within the meaningof the invention when the difference to those measured for the T26Ssubstitution under essentially the same experimental conditions is lessthan 2 fold, preferably less than 1.5 fold or even less than 1.2 fold.

Generally, within the meaning of the invention, lentiviruses areexogenous, non-oncogenic retroviruses and include, but are not limitedto, equine infectious anemia virus (EIAV; U.S. Pat. No. 6,277,633),simian immunodeficiency viruses (SIVs), visna and progressive pneumoniaviruses of sheep, feline immunodeficiency virus (FIV), bovineimmunodeficiency virus (BIV) and human immunodeficiency viruses (HIV-1and HIV-2).

The lentiviral genome includes three genes found in retroviruses: gag,pol and env, which are flanked by two long terminal repeat (LTR)sequences. The gag gene encodes the internal structural proteins, suchas matrix, capsid and nucleocapsid proteins; the pol gene encodes theRNA-directed DNA polymerase (reverse transcriptase (RT)), a protease andan integrase; and the env gene encodes viral envelope glycoproteins. The5′ and 3′ LTR's serve to promote transcription and polyadenylation ofthe virion RNA's. The LTR contains all other cis-acting sequencesnecessary for viral replication. Lentiviruses may have additional genesincluding vif, vpr, tat, rev, vpu, nef and vpx (in HIV-1, HIV-2 and/orSIV). Adjacent to the 5′ LTR are sequences necessary for reversetranscription of the genome, such as the tRNA primer binding site, andfor efficient encapsidation of viral RNA into particles, such as the Psisite. If the sequences necessary for encapsidation are missing from theviral genome, such a cis defect will prevent encapsidation of genomicRNA. However, the resulting mutant remains capable of directing thesynthesis of all virion proteins.

In one embodiment of the invention the packaging construct of theinvention comprises a lentiviral gag gene. The gag gene is the 5′-mostgene on retroviral genomes and, as has been described in more detailabove, encodes structural proteins that are required to form the virusparticle. The gag gene is translated to give a precursor polyproteinthat is subsequently cleaved to yield three to five structural proteins.In a preferred embodiment, the gag gene is recoded.

A gene that is “recoded” refers to a gene or genes that are altered insuch a manner that the polypeptide encoded by a nucleic acid remains thesame as in the unaltered sequence but the nucleic acid sequence encodingthe polypeptide is changed. It is well known in the art that due todegeneracy of the genetic code, there exist multiple DNA and RNA codonswhich can encode the same amino acid translation product. For example,in one embodiment, a DNA sequence encoding the gag and or pol genes ofBIV is “recoded” so that the nucleotide sequence is altered but theamino acid translation sequence for the GAG and POL polypeptides remainidentical to the wildtype amino acid sequence. Furthermore, it is alsoknown that different organisms have different preferences forutilization of particular codons to synthesize an amino acid. Recodingcan be accomplished using various techniques known in the art ormodification thereof, for example, as taught in Casimiro D. R., WrightP. E., Dyson H. J., “PCR-based gene synthesis and protein NMRspectroscopy”, Structure, 1997 Nov. 15, 5(11):1407-12; Brocca S.,Schmidt-Dannert C., Lotti M., Alberghina L., Schmid R. D., “Design,total synthesis, and functional overexpression of the Candida rugosalip1 gene coding for a major industrial lipase”, Protein Sci., 1998June, 7(6):1 415-22 Related Articles, Books, LinkOut Design;Withers-Martinez C., Carpenter E. P., Hackett F., Ely B., Sajid M.,Grainger M., Blackman M. J., “PCR-based gene synthesis as an efficientapproach for expression of the A+T-rich malaria genome”, Protein Eng.,1999 December, 12(12):1113-20; Stemmer W. P., Crameri A., Ha K. D.,Brennan T. M., Heyneker H. L., “Single-step assembly of a gene andentire plasmid from large numbers of oligodeoxyribonucleotides”, Gene,1995 Oct. 16, 164(1):49-53.

In one preferred embodiment of the present invention the packagingconstruct of the invention is derived from the HIV genome. In aparticularly preferred embodiment the packaging construct furthercomprises a mutation in a HIV vif or vpr gene. Further particularlypreferred embodiments of the present invention are the pΔVΔR-PR*construct and the pΔVΔR-PR*SAR construct as described in the Exampleshereinbelow.

In one preferred embodiment the packaging construct is derived from theBIV genome. The basic genomic organization of BIV is disclosed in Garveyet al., (Virology, 175:391-409, 1990) and U.S. Pat. No. 5,380,830.Additionally disclosed are methods of obtaining BIV genomic DNA from BIVinfected cells. Sequences encoding BIV and plasmids containingretroviral genomes suitable for use in preparing the vector constructsmay be readily obtained given the disclosure provided herein or fromdepositories and databases such as the American Type Culture Collection(ATCC), for example, ATCC Accession No. 68092 and ATCC Accession No.68093 and GENBANK. BIV based vectors are described in PCT Publication WO01/44458.

The gag and pol genes are in different frames and overlap. The pol andenv genes are in the same reading frame and are separated by the“central region”. There are five open reading frames (ORFs) found in thecentral region. Three of these are similar in structure to the exons forvif, tat and rev of HIV and other lentiviruses. The other two ORFs arelocated in a position in the central region analogous to vpr, vpx andvpu encoding ORFs of HIV-1 and/or HIV-2. The nef ORF which is locatedpost-env in the genomes of other lentiviruses appears to be lacking inBIV.

It will be understood that for the nucleotide sequence of the BIVgenome, natural variations can exist between individual BIV viruses.These variations may result in deletions, substitutions, insertions,inversions or additions of one or more nucleotides as long as theclaimed function of the gene is not lost. The DNA sequences encodingsuch variants may be created by standard cloning methods or polymerasechain reaction (PC R), see U.S. Pat. Nos. 4,683,195 and 4,683,202. Thepresent invention relates to a nucleic acid segment from a BIV genomeobtainable from any strain or clone of BIV. In one embodiment of thisinvention, the BIV vector construct of the invention includes asufficient number of nucleotides corresponding to nucleotides of the BIVgenome to express one or more functional BIV genes.

In a preferred embodiment the BIV-derived packaging construct of theinvention may comprise a mutation in, including deletion of all or aportion of, a BIV vif, W, Y or tat gene. The BIV Rev gene andRev-responsive element (RRE) may also be mutated or deleted ifConstitutive Transport Element (CTE) is used in the BIV vector of theinvention.

In a further embodiment of the invention, the lentiviral vector of thepresent invention comprises a DNA scaffold attachment region (SAR),which as broadly defined herein, refers to a DNA sequence having anaffinity or intrinsic binding ability for the nuclear scaffold ormatrix. Particularly preferred is an IFN-SAR element and most preferredis a β-IFN-SAR element. SAR elements are usually 100 to 300 or more basepairs long, and may require a redundancy of sequence information andcontain multiple sites of protein-DNA interaction. SAR elements are DNAelements which bind to the isolated nuclear scaffold or matrix with highaffinity (Cockerill, P. N. and Garrard, W. T. (1986). Cell 44: 273-282,Gasser, S. M. and Laemmli, U. K. (1986). Cell 46: 521-530). Some of theSAR sequences have been shown to have enhancer activities (Phi-Van, L.,et al (1990). Mol. Cell Biol. 10: 2302-2307, McKnight, R. A., et al.(1992). Proc. Natl. Acad. Sci. USA 89: 6943-6947), and some serve ascis-acting elements, driving B-cell specific demethylation in theimmunoglobulin k locus (Lichtenstein, M. et al., (1994). Cell 76:913-923, Kirillov, A. et al., (1996). Nat. Genet. 13: 435-441). ThehIFN-β SAR element inhibits de novo methylation of the 5′ LTR, andappears to insulate the transgene from the influence of the flankinghost chromatin at the site of retroviral integration. Position effectsare thus decreased. SAR elements may be obtained, for example, fromeukaryotes including mammals, plants, insects and yeast, preferablymammals. Examples of suitable protocols for identifying SAR elements foruse in the present invention are described in WO96/19573.

Preferably the SAR elements should be located downstream from thetransgene and the lentiviral env sequence. In one embodiment, more thanone SAR element may be inserted into the packaging vector of theinvention. Although Applicants do not wish to be bound by mechanisticspeculation, the use of flanking SAR elements in the nucleic acidmolecules may allow the SAR elements to form an independent loop orchromatin domain, which is insulated from the effects of neighboringchromatin.

Other methods may be used in addition or as an alternative to using SARelements. These methods include integrating the gag/pol expressionconstruct in a highly expressed region of a chromosome or a highlyexpressed gene. These highly expressed regions include, but are notlimited to, SARs, locus control regions (LCRs), and insulator regions(Emery, et al., PNAS, 97(16):9150-9155 (2000)). It will be evident toone skilled in the art that there are several methods which can beemployed to integrate a gag/pol expression construct into a highlyexpressed region or gene (e.g., homologous recombination).

In a further aspect of the present invention there is provided a stablepre-packaging cell line comprising the packaging construct of theinvention. Particularly preferred pre-packaging cell lines are such celllines which are capable of stably expressing at least 5 ng/ml of the HIVp24 protein, or at least 5 ng/ml of BIV reverse transcriptase (RT)protein, and wherein such protein expression is constitutive.Preferably, 50 ng/ml of BIV RT is produced. More preferably, 500 ng/mlBIV RT is produced.

In a further aspect of the present invention there is provided a stablepackaging cell line comprising the packaging construct of the inventionand further comprising a plasmid comprising an env gene. Accordingly, a“packaging cell line” within the meaning of the invention is arecombinant cell line containing nucleic acid sequences expressingretroviral Gag, Pol and Env structural proteins. Because the packagingcell line lacks the retroviral nucleic acid sequence of the packagingsignal and other cis-acting elements, infectious virions cannot beproduced.

The “env” gene encodes the envelope proteins. As used in thisdisclosure, the env gene includes not only natural env gene sequencesbut also modifications to the env gene including modifications thatalter target specificity of retroviruses and lentiviruses or env genesthat are used to generate pseudotyped retrovirus/lentivirus, referenceis made to PCT Publications WO 92/14829, WO 94/11524, and U.S. Pat. No.6,004,798. The env gene can be derived from any virus, includingretroviruses. The env preferably is an amphotropic envelope proteinwhich allows transduction of cells of human and other species. It may bedesirable to target the recombinant virus by linkage of the envelopeprotein with an antibody or a particular ligand for targeting to areceptor of a particular cell-type. By inserting a sequence including aregulatory region of interest into the viral vector, along with a genewhich encodes the ligand for a receptor on a specific target cell thevector may be rendered target-specific. For example, vectors can be madetarget-specific by inserting, for example, a glycolipid or a protein.Further, targeting may be accomplished by using an antigen-bindingportion of an antibody or a recombinant antibody-type molecule, such asa single chain antibody, to target the retroviral vector. The personskilled in the art will know of, or can readily ascertain without undueexperimentation, specific methods to achieve delivery of a retroviralvector to a specific target.

Generally, the cell lines of the invention may include separate vectorswhich provide the packaging functions of recombinant virions, such as,gag, pol, env, tat and rev, as discussed above. There is no limitationon the number of vectors which are utilized so long as the vectors areused to transform and to produce the packaging cell line to yieldrecombinant lentivirus. The vectors are introduced via transfection orinfection into the packaging cell line. The packaging cell line producesviral particles that contain the vector genome. Methods for transfectionor infection are well known by those of skill in the art. Aftercotransfection of the packaging vectors and the transfer vector to thepackaging cell line, the recombinant virus is recovered from the culturemedia and titered by standard methods used by those of skill in the art.Thus, the packaging constructs can be introduced into human cell linesfor example by calcium phosphate transfection, lipofection orelectroporation, generally together with a dominant selectable marker,such as neo, DHFR, Gln synthetase or ADA, followed by selection in thepresence of the appropriate drug and isolation of clones.

In a preferred embodiment the packaging cell line of the inventionincludes the VSV-G env gene. While VSV-G protein is a desirable env genebecause VSV-G confers broad host range on the recombinant virus, VSV-Gcan be deleterious to the host cell. Thus, when a gene such as that forVSV-G is used, it is preferred to employ an inducible promoter system sothat VSV-G expression can be regulated to minimize host toxicity whenVSV-G expression is not required. For example, thetetracycline-regulatable gene expression system of Gossen & Bujard(Proc. Natl. Acad. Sci. (1992) 89:5547-5551) can be employed to providefor inducible expression of VSV-G. The tet/VP16 transactivator may bepresent on a first vector and the VSV-G coding sequence may be cloneddownstream from a promoter controlled by tet operator sequences onanother vector. Other non-limiting examples of regulatable expressionsystems are described in PCT Publications WO 01/30843 and WO 02/06463.

In another preferred embodiment, the packaging cell line of theinvention includes the LCMV mutant env gene (Beyer, et al., J. Virol.,76:1488-1495). In one embodiment, the LCMV mutant env gene isconstitutively expressed. In another embodiment, the LCMV mutant envgene is expressed from an inducible promoter. Inducible promoter systemsare described hereinabove.

In a further aspect of the present invention there is provided aproducer cell line comprising the packaging construct of the invention,an env gene and further comprising a lentiviral vector, i.e. a vectorcomprising a lentiviral 5′LTR, a lentiviral 3′LTR and a suitablepackaging signal. Accordingly, a “producer cell line” is a packagingcell line as defined above which also contains a replication-defectivelentiviral vector which is packaged into the vector particle. Theproducer cell produces lentiviral-based particles, which may contain“heterologous” (i.e., non-lentiviral) genes, such as therapeutic ormarker genes.

In a preferred embodiment the producer cell line of the invention isfurther characterized in that it is capable of producing a lentiviralvirus titer of at least 10E5 cfu/ml and preferably ≧10 E6 cfu/ml.

In yet another aspect a lentiviral vector particle obtained from thestable producer cell line of the invention is provided. Also provided isa method for producing a lentiviral vector particle preparationcomprising the steps of transfecting the stable packaging cell line ofthe invention with a lentiviral vector, isolating and propagating aproducer cell line in a suitable culture medium and obtaining alentiviral vector particle preparation from the said culture medium.

Generally, viral supernatants are harvested using standard techniquessuch as filtration of supernatants at an appropriate time-point, such asfor example 48 hours after transfection. The viral titer is determinedby infection of suitable cells with an appropriate amount of viralsupernatant. For example, forty-eight hours later, the transductionefficiency is assayed. Thus, the instant invention provides methods andmeans for producing high titer recombinant lentiviral vector particles.Such particle preparations can subsequently be used to infect targetcells using techniques known in the art.

The following examples are provided for the purpose of furtherillustrating the present invention but are in no way to be taken aslimiting.

EXAMPLES Example 1 HIV-Based Packaging Vector Construction

The packaging plasmids used in this study are depicted in FIG. 1A.pHIVΔΨ contains the sequence of the HIV-1 NL4-3 isolate with deletionsof 1) both LTRs, 2) 33 bp of the packaging signal (Ψ) 5′ to the gaggene, 3) 1587 bp of the env gene, 4) the vpu gene and 5) the nef gene.All the other genes are unaffected. Transcription of the HIV genes isunder the control of the Cytomegalovirus (CMV) promoter, derived fromthe pCI vector (Promega, Wis.). pHIVΔΨ is a modification of pHIV-PV(Sutton, R. E., H. T. M. Wu, R. Rigg, E. Bohnlein, and P. O. Brown.1998. 72:5781-5788). A 660 bp NdeI/SalI fragment was deleted from pHIVΔΨto remove vif and vpr, resulting in pΔVΔR. A point mutation (nt 2328, Ato T)) was introduced into the protease gene of pΔVΔR using PCRmutagenesis to make pΔVΔR-PR*. This corresponds to an amino acidsubstitution of Thr26 to Ser26. The PCR primers for the mutagenesis areas follows: Primer A: 5′-AATTGCAGGGCCCCTAGGAAAAA-3′ (SEQ ID NO:5) PrimerB: 5′-TCTGCTCCTGA ATCTAATAGCGCTT-3′ (SEQ ID NO:6) Primer C:5′-AAGCGCTATTAGATTCAGGAGCAGA-3′ (SEQ ID NO:7) Primer D:CCATGTACCGGTTCTTTTAGAATC-3′. (SEQ ID NO:8)

Primers B and C are complementary and contain the A to T mutation (nt2328), which confers the Thr to Ser amino acid substitution and a T to Gmutation (nt 2318), which introduces a unique Eco47III restriction site,but does not alter the amino acid sequence. The PCR product amplifiedfrom primers A & B was purified and combined with the purified productamplified from primers C & D. This mix was then amplified with primers A& D, cut with ApaI and AgeI (restriction sites naturally present in theprimers) and then cloned back into pΔVΔR. The presence of the mutationwas confirmed by Eco47II digestion and sequence analysis. The interferonβ scaffold attachment region (SAR) (800 bp fragment) (Agarwal, M., T. W.Austin, F. Morel, J. Chen, E. Böhnlein, and I. Plavec. 1998.72:3720-3728) was introduced into a NotI restriction site (nt 8800) forboth pΔVΔR and pΔVΔR-PR* to create pΔVΔR-SAR and pΔVΔR-PR*SAR,respectively. The transfer vector used in these studies, pHLEIP, isshown in FIG. 1B. pHLEIP contains sequences from the HIV-1 NL4-3 isolateincluding 1) both LTRs, 2) 1251 bp of the 5′ end of gag, 3) 715 bp ofthe 3′ end of pol, which contains the central polypurine tract (ppt) andtranscriptional enhancer sequences, 4) 311 bp encoding the first exonsof tat and rev, and 5) 977 bp of env containing the REV response element(RRE) and the second exon of tat. The nef and rev coding sequences aredisrupted by the insertion of the egfp marker gene (Clontech, CA.),followed by the picornoviral internal ribosomal entry site (Jang, S. K.,M. V. Davies, R. J. Kaufman, and E. Wimmer. 1989. J. Virol.63:1651-1660) and the puromycin N-acetyltransferase gene (Vara, J. A.,A. Portela, J. Ortin, and A. Jimenez. 1986. Nuc. Acids Res.14:4617-4624). The expression of egfp is controlled by the HIV LTR in atat-dependent manner. This vector is a modification of pHIV-AP G⁻P⁻E⁻F⁻V(Sutton, R. E., H. T. M. Wu, R. Rigg, E. Bohnlein, and P. O. Brown.1998. Journal of Virology. 72:5781-5788). The envelope constructs usedfor pseudotyping are pCIGL, which contains the VSV-G gene (Burns, J. C.,T. Friedmann, W. Driever, M. Burrascano, and J.-K. Yee. 1993.Proceedings of the National Academy of Sciences, USA. 90:8033-8037; Yee,J. K., A. Miyanohara, P. LaPorte, K. Bouic, J. C. Burns, and T.Friedmann. 1994. Proceedings of the National Academy of Sciences USA.91:9564-9568) under the control of the CMV promoter from pCI, andpCMV*Ea, which contains the amphotropic murine leukemia virus (A-MLV)envelope gene cloned into pCI (Rigg, R. J., J. Chen, J. S. Dando, S. P.Forestell, I. Plavec, and E. Bohnlein. 1996. Virology. 218:290-295)(FIG. 1C).

Example 2 Functional Analysis of Packaging Constructs

A transient assay was performed to verify that the packaging constructsretained all necessary functions. First viral supernatants weregenerated by transfecting 293T cells (5×10⁶) with 3 constructs (10 μgpackaging construct, 5 μg envelope construct, and 20 μg transfervector), by Ca₂PO₄ precipitation (Clontech, CA.). The transfectionsupernatants were collected after 24, 48 and 72 hours, pooled andfiltered through a 0.45 μm filter. To determine transductionefficiencies, the collected vector supernatants were diluted 1:1 withculture medium (DMEM plus 10% FBS), added to 2×10⁵ 293T cells plated ona 6 well dish, and centrifuged at 2500×g in the presence of 8 μg/mlprotamine sulfate (Sigma, MO.). This transduction protocol known as“spinoculation” (Bahnson, A. B., J. T. Dunigan, B. E. Baysal, T. Mohney,R. W. Atchison, M. T. Nimgaonkar, E. D. Ball, and J. A. Barranger. 1995,J Virol Meth. 54:131-143) was performed at 37° C. for 3-4 hours. Afterspinoculation, the medium was replaced and the cells were cultured at37° C. for 48-72 hr. After incubation, the cells were fixed in 1-2%formaldehyde and EGFP expression was measured by flow cytometry on aFACScan (Becton Dickinson, MD.).

Example 3 Production of Producer Cell Lines

To generate Gag/Pol producer cell lines, 293 Ea6 cells (5×10⁶) wereplated in a 10 cm dish and transfected with 2 constructs (10 μgpackaging construct and 1 μg pcDNApuro) by Ca₂PO₄ precipitation(Clontech, CA.). PCDNApuro is a plasmid containing the puromycinN-acetyltransferase gene driven by the CMV promoter from pCDNA1.1/Amp(Invitrogen, CA.). The 293 Ea6 cell line constitutively expresses theA-MLV envelope (Rigg, R. J., J. Chen, J. S. Dando, S. P. Forestell, I.Plavec, and E. Bohnlein. 1996. Virology. 218:290-295). Aftertransfection, the cells were cultured for 48 hrs and then transferred tomedium containing 5 μg/ml puromycin (Sigma, MO.). The cells weremaintained under puromycin selection and monitored periodically for Gagproduction via p24 ELISA (Beckman/Coulter, CA.). To measure p24production of the cell line, 1×10⁶ cells were plated in a well of a6-well dish, supernatant was collected 24 hr post plating, filteredthrough a 0.45 μn filter, and then assayed by p24 ELISA. Single cellclones were obtained from the cell line expressing the highest level ofp24 by sorting on a FACStar (Becton Dickinson, MD.). The transfervector, pHLEIP was introduced into the clone with the highest stableproduction of p24. This was achieved by transducing the clone withtransient VSV-G pseudotyped pHLEIP vector supernatant as described inthe previous section. Supernatants from the resulting packaging linewere collected at various times post transduction and titered on 293Tcells. For titering, 2×10⁵ 293T cells were plated in wells of a 6-welldish and transduced as previously described with 10 fold serialdilutions of viral supernatant. After 48 hr of culture, 5 μg/mlpuromycin was added to the medium for selection. The cells were thenmaintained in selection medium for 2 weeks. The surviving colonies werefixed and stained in coomassie blue solution (50% methanol, 0.05%coomassie brilliant blue R-250, 10% acetic acid) and counted todetermine the titer.

Example 4 Evaluation of Packaging Constructs for Stable p24 Production

To determine if any of the modified packaging constructs could conferlong-term, high-level Gag/Pol protein expression, stable 293 Ea6-basedcell lines were generated and monitored for viral particle production atregular intervals after selection as described in Materials and Methods.FIG. 2 is a graph comparing the viral production, as measured by p24levels, from the different packaging cell lines over time (approximately12 weeks). As expected, the cells containing the construct with vpr,pHIVΔΨ, expressed low levels of p24 soon after selection and by thesecond passage the p24 expression was below the level of detection. Thecell line made with the vpr-deleted construct, pΔVΔR and the two celllines containing either the protease mutation, pΔVΔR-PR* or the SARinsertion, pΔVΔR-SAR had higher initial p24 levels (3-12 ng/ml), butdecreased to levels less than 1 ng/ml. Interestingly, the cell linecontaining the construct with both the protease mutation and the SARinsertion, pΔVΔR-PR*SAR, maintained about 4 times more p24 expressionthan the cells containing the single modification constructs. Theseresults suggest that inhibiting both protease toxicity and promotersilencing can increase the levels and stability of p24 expression in acell line, but that either modification alone provides no significantimprovement.

Example 5 Clonal Analysis of Modified Packaging Constructs

Although the cell line containing the double modified construct,pΔVΔR-PR*SAR expressed the highest levels of p24 compared to the otherconstructs, these levels are not sufficient to generate an efficientpackaging cell line. Single cell clones were isolated from this cellline in an attempt to find a high p24 producing clone. As shown in Table1 the majority of clones expressed negligible levels of p24, but 2 ofthe 40 clones analyzed expressed significantly higher levels of p24 thanthe parent cell line (10-100 ng/ml). One of these clones (PR*SAR clone)expressed 100 ng/ml p24 for at least 12 weeks. To verify the importanceof the protease mutation for allowing high-level p24 production, singlecell clones were also obtained from the cell line containing pΔVΔR-SARand analyzed for p24 production. Table 1 illustrates the comparisonbetween clones containing the packaging constructs+/−the proteasemutation. No pΔVΔR-SAR containing clones expressed p24 levels >10 ng/mleven though almost twice as many clones were evaluated compared to thosecontaining pΔVΔR-PR*SAR. These results confirm the importance of theprotease mutation in producing high-level Gag/Pol producer cells. TABLE1 Clonal analysis confirms the importance of the protease mutation inobtaining a high-level Gag producing cell line p24 Production (ng/ml)Background Total # of Cell line Level* 1-10 10-100 clones ΔVΔR-PR*SAR 335 2 40 ΔVΔR-SAR 71 5 0 76*Background Level is <20 pg/ml p24

Example 6 Titer of Highest Gag/Pol-Producing Packaging Cell Clone

To determine how efficiently the PR*SAR clone could package and transfervector, a titration analysis was performed. A transiently produced VSV-Gpseudotyped vector containing EGFP and a puromycin resistance gene,pHLEIP was introduced into the packaging cell clone via transduction, asdescribed in Materials and Methods. Supernatants were collected atvarious time points post transduction, analyzed for p24 production, andtitered on 293T cells via EGFP FACS and puromycin selection. As shown inTable 2 Expt. 1, the p24 production at the 24 hr time point was 91 ng/mland the titers of supernatant collected at both 24 and 48 hours were5×10⁴ IU/ml as determined by puromycin selection. Expt. 2 was similar toexpt. 1 except the virus was allowed to accumulate over the indicatedcollection times before analysis. Under these conditions, the p24production went from 954 ng/ml at 48 hr to 2300 ng/ml by 96 hr. Theselevels are now in the range produced by the transient system (1-10μg/ml) (data not shown). Interestingly, although the p24 levelsincreased with accumulation, the supernatants from all three time-pointshad similar titers (4-6×10⁴ IU/ml) on 293T cells. This corresponded to<1% EGFP expression in 293T cells transduced with the 48 hr and 72 hraccumulated supernatants. Also, while the viral supernatants generatedfrom the transient system have p24 levels corn parable to the PR*SARclone after accumulation, they routinely have titers of 5-10×10⁶ IU/ml(Table 3). This data suggests that p24 production is probably not thelimiting factor in achieving high titers from the packaging clone. TABLE2 p24 production and titer of PR*SAR clone p24 Titer IU/ml SupernatantProduction (Puromycin % EGFP Experiment Collection (ng/ml) Selection)(FACS) Expt. 1 24 hr  91  5 × 10⁴ ND 48 hr ND  5 × 10⁴ ND Expt. 2 48 hr 954 4-6 × 10⁴ 0.90 accumulation 72 hr 1800 4-6 × 10⁴ 0.80 accumulation96 hr 2300 4-6 × 10⁴ ND accumulation

Example 7 Effect of Envelope Expression on Titer of Packaging Cell Clone

To determine if a loss of envelope expression could be contributing tothe lower titers of the PR*SAR packaging clone, first a FACS analysiswas performed to verify A-MLV env expression. An equivalent level ofenvelope was detectable by FACS compared to the 293 Ea6 parent cellline. To further test whether the envelope was limiting,envelope-expressing constructs were transfected into the PR*SARpackaging clone, which had already been stably transduced with thepHLEIP vector. Both VSV-G and A-MLV env expression constructs were used.Table 3 shows a comparison of the titers and transduction efficienciesof supernatant from the stable packaging clone in the presence orabsence of additional envelope. The addition of VSV-G increased thetiter 5-8 fold and allowed for a detectable transduction efficiency of14%. The addition of A-MLV env also increased the titer, but only 2fold. These results indicate that the titer of the PR*SAR packaging cellclone can be improved by increasing envelope expression.

The levels of Gag produced from our PR*SAR packaging clone, reach thelevels obtained with the transient packaging system. However, the titersare still lower than with the transient system. We have shown thatenvelope is limiting in the clone we isolated, therefore screening moreclones based on envelope expression as well has p24 production mightincrease the probability of obtaining a higher titer clone. In addition,the transfer vector was introduced into the packaging clone by only oneround of transduction, thereby limiting the vector copy number.Increasing the vector copies in the packaging cell line should alsoimprove titers. TABLE 3 The titer and transduction efficiency of thePR*SAR clone is improved with increased envelope expression Titer IU/mlPackaging Added (Puromycin % EGFP System Envelope Selection) (FACS)Stable None 4-6 × 10⁴  <1 A-MLV 1 × 10⁵ ND VSV-G 3 × 10⁵ 14 TransientA-MLV 5 × 10⁶ 22 VSV-G 2 × 10⁷ 91

Example 8 Construction of Packaging Constructs for BIV Based LentiviralVectors

To generate a BIV based lentiviral packaging construct, CTE is P CRamplified with two primers CTE1 (5′-CGGGGTACCACCTCCCCTGTGAGCTAG-3′) (SEQID NO:9) and CTE2 (TGCTCTAGAGACACATCCCTCGGAGGC-3′) (SEQ ID NO:10). Theamplified product is digested with KpnI and XbaI and ligated to a pClplasmid previously digested with KpnI and XbaI, generating pCl.CTE.Second, BIV gag and pol coding sequence is PCR amplified with twoprimers GAG5 (5′-CCGCTCGAGATGAAGAGAAGGCGAGTTAGAA-3′) (SEQ ID NO:11) andPOL3 (5′-CCGCTCGAGTCACGAACTCCCATCTTGGAT-3′) (SEQ ID NO:12). Theamplified product is digested with XhoI and ligated to pCl.CTEpreviously digested with XhoI, generating a BIV based packagingconstruct, pClBIVGP. Alternatively, CTE can be replaced by BIV RRE(Rev-responsive element) and Rev. To create the Threonine to Serine inBIV protease (corresponding to amino acid number 26 from the start ofpProtease) to generate a potentially less toxic BIV protease, pCIBIVGPis subjected to PCR amplification with primer Primer A(5′-GGGTTAGTAGACTCTGGA-3′) (SEQ ID NO:13) and Primer B(5′-GCCCGGGTCGACTCTAGA-3′) (SEQ ID NO:14). Primer B contains the A to Tmutation, which confers the Thr to Ser amino acid substitution. The PCRproduct amplified from primers A and B is digested with AccI and ligatedto pCIBIVGP previously digested with AccI resulting in pCIBIVGPmut withthe desired mutation in the protease.

Example 9 BIV Packaging Constructs with Recoded gag/pol Sequence orRecoded gag/pol Sequence with Specific Mutation in Protease

Without being bound by theory, lentiviruses such as HIV, SIV and BIV arethought to contain nucleic acid sequences in their viral RNAs whichcause RNA instability, thereby preventing efficient nuclear export ofviral RNAs. This is believed to be due to the fact that lentivirusesemploy rare codon usage and/or RNA secondary structure which isdetermined by the RNA sequence. The viral RNAs containing these rarecodons can not be efficiently transported out of the nucleus withoutRev/RRE. We recoded the BIV gag/pol coding sequence using preferred Homosapiens codons (Table 4) to eliminate RRE from the packaging construct,to minimize or eliminate the overlaps between the packaging and transfervector constructs and to increase the BIV gag/pol gene expressionlevels. The sequence as in SEQ ID NO:15 was selected for the recodedgag/pol construct. The company Aptagen (Herdon, VA.) was contracted toclone this DNA construct. The recoded gag/pol coding sequence was clonedinto the pCI mammalian expression vector, generating pCligpSyn (FIG. 3).The generation of pCligpSyn allowed us to produce BIV vectors from afour component system by cotransfecting pCligpSyn, pTracerARev (a BIVRev expression construct containing SEQ ID NO:16; Table 7), pBIVminivec(a BIV-based transfer vector construct encoding GFP), and pCMVVSV-G (aVSV-G expression construct). The BIV vectors generated from this systemwith recoded gag/pol were fully functional as indicated by their abilityto efficiently transduce cells (Table 5). TABLE 4 Sequence of recodedgag/pol (SEQ ID NO:15)ATGAAGCGGAGAGAGCTGGAGAAGAAACTGAGGAAAGTGCGCGTGACACCTCAACAGGACAAGTACTATACCATCGGCAACCTGCAGTGGGCCATCCGCATGATCAACCTGATGGGCATCAAGTGCGTGTGCGACGAGGAATGCAGCGCCGCTGAGGTCGCCCTGATCATCACCCAGTTTAGCGCGCTCGACCTGGAGAACTCCCCTATCCGCGGCAAGGAAGAGGTGGCCATCAAGAATACCCTGAAGGTGTTTTGGAGCCTGCTGGCCGGATACAAGCCTGAGAGCACCGAGACCGCCCTGGGATACTGGGAAGCCTTCACCTACAGAGAGAGGGAAGCTAGAGCCGACAAGGAGGGAGAGATCAAGAGCATCTACCCTAGCCTGACCCAGAACACCCAGAACAAGAAACAGACCAGCAATCAGACAAACACCCAGAGCCTGCCCGCTATCACCACACAGGATGGCACCCCTCGCTTCGACCCCGACCTGATGAAGCAGCTGAAGATCTGGTCCGATGCCACAGAGCGCAATGGAGTGGACCTGCATGCCGTGAACATCCTGGGAGTGATCACAGCCAACCTGGTGCAAGAAGAGATCAAGCTCCTGCTGAATAGCACACCCAAGTGGCGCCTGGACGTGCAGCTGATCGAGAGCATAAGTGAGAGAGAAGGAGAACGCCCACCGCACCTGGAAGCAGCATCACCCTGAGGCTCGCAAGACAGACGAGATCATTGGAAAGGGACTGAGCTCCGCCGAGCAGGCTACCCTGATCAGCGTGGAGTGCAGAGAGACCTTCCGCCAGTGGGTGCTGCAGGCTGCCATGGAGGTCGCCCAGGCTAAGCACGCCACACCCGGACCTATCAACATCCATCAAGGCCCTAAGGAACCCTACACCGACTTCATCAACCGCCTGGTGGCTGCCCTGGTAAGGAATGGCCGCTCCCGAGACCACAAAGGAGTACCTCCTGCAGCACCTGAGCATCGACCACGCCAACGAGGACTGTCAGTCCATCCTGCGCCCTCTGGGACCCAACACACCTATGGAGAAGAAACTGGAGGCCTGTCGCGTGGTGGGAAGCCAGAAGAGCAAGATGCAGTTCCTGGTGGCCGCTATGAAGGAAATGGGGATCCAGTCTCCTATTCCAGCCGTGCTGCCTCACACACCCGAAGCCTACGCCTCCCAAACCTCAGGGCCCGAGGATGGTAGGAGATGTTACGGATGTGGGAAGACAGGACATTTGAAGAGGAATTGTAAACAGCAAAAATGCTACCATTGTGGCAAACCTGGCCACCAAGCAAGAAACTGCAGGTCAAAAAACGGGAAGTGCTCCTCTGCCCCTTATGGGCAGAGGAGCCTAACCACAGAACAATTTTCACCAGAGCAACATGAGTTCTGTGACCCCATCTGCACCCCCTCTTATATTAGATTAGACAAACAGCCTTTTATAAAGGTGTTCATTGGCGGCCGCTGGGTGAAGGGACTGGTGGACACAGGCGCTGACGAGGTGGTGCTGAAGAACATCCACTGGGACCGCATCAAAGGCTACCCTGGAACACCCATCAAGCAGATCGGCGTGAACGGCGTGAACGTGGCTAAGCGCAAAACACATGTGGAGTGGAGATTCAAAGACAAGACCGGCATCATTGACGTCCTCTTCAGCGACACACCTGTGAACCTGTTTGGCAGAAGCCTGCTCAGATCCATCGTGACCTGCTTTACCCTGCTGGTGCACACCGAGAAGATCGAGCCACTGCCTGTGAAGGTGCGCGGCCCTGGACCTAAGGTGCCACAATGGCCCCTGACCAAGGAGAAATACCAGGCCCTGAAGGAGATCGTGAAGGACCTGCTGGCCGAGGGAAAGATCAGCGAAGCTGCCTGGGACAACCCTTACAACACACCCGTGTTCGTGATCAAGAAGAAAGGCACCGGCCGCTGGCGCATGCTGATGGACTTCCGCGAGCTGAATAAGATCACCGTGAAAGGCCAAGAGTTCAGCACAGGACTCCCTTATCCACCCGGCATCAAGGAGTGTGAGCACCTGACCGCCATCGACATCAAGGACGCCTACTTCACCATCCCTCTGCACGAGGACTTCAGACCCTTCACAGCCTTCAGCGTGGTCCCAGTGAACCGCGAGGGCCCCATCGAGCGCTTCCAGTGGAACGTCCTGCCTCAAGGCTCAAGGTGTGCTCCCCTGCCATCTACCAGACCACAACCCAGAAGATCATTGAGAACATCAAGAAGAGCCATCCCGACGTGATGCTGTATCAGTACATGGATGACCTCCTGATTGGCAGCAATCGCGATGACCACAAGCAGATCGTGCAGGAGATCAGAGACAAGCTGGGCAGCTATGGCTTCAAGACACCCGACGAGAAAGTGCAGGAAGAGCGCGTGAAGTGGATCGGCTTCGAGCTGACACCTAAGAAATGGAGATTCCAGCGTAGGCAACTGAAGATCAAGAACCCACTGACCGTGAACGAACTCCAGCAGCTGGTCGGCAACTGTGTGTGGGTGCAGCCCGAGGTGAAGATCCCTCTGTACCCACTGACCGATCTGCTCCGCGACAAGACCAACCTGCAGGAAAAGATCCAGCTGACACCCGAGGCCATCAAGTGCGTGGAAGAGTTCAACCTGAAGCTGAAAGATCCCGAGTGGAAGGACAGAATTCGCGAAGGAGCCGAGCTGGTGATCAAGATCCAAATGGTCCCTCGCGGCATCGTGTTCGACCTGCTGCAAGACGGCAATCCTATCTGGGGAGGCGTGAAAGGACTGAACTACGACCACAGCAACAAGATCAAGAAGATCCTGCGCACCATGAACGAGCTGAACCGCACCGTGGTGATCATGACCGGACGCGAAGCTAGCTTTCTCCTGCCTGGATCCAGCGAGGATTGGGAGGCCGCCCTGCAGAAGGAGAGAGCCTGACCCAAATCTTTCCCGTGAAGTTCTACCGCCATAGCTGTAGATGGACAAGCATCTGTGGACCCGTCCGCGAGAACCTGACCACCTACTATACCGACGGCGGGAAGAAAGGAAAGACAGCTGCCGCAGTGTACTGGTGTGAAGGAAGAACTAAGAGCAAAGTGTTCCCTGGAACCAATCAACAGGCTGAGCTGAAGGCAATCTGCATGGCTCTGCTGGACGGACCTCCCAAGATGAACATCATCACCGACAGCCGCTACGCTTATGAGGGCATGAGAGAGGAACCTGAGACCTGGGCTCGCGAGGGCATCTGGCTGGAGATTGCAAAGATCCTGCCATTCAAGCAATACGTCGGAGTGGGCTGGGTCCCTGGTCACAAAGGCATTGGAGGCAATACCGAGGCTGACGAAGGAGTGAAGAAAGCCCTGGAGCAAATGGCACCATGTTCCCCTCCCGAGGCTATCCTGCTCATAAACCTGGCGAGAAGCAAAACCTGGAGACCGGCATCTACATGCAAGGCCTGAGACCTCAGAGCTTCCTGCCCCGCGCTGACCTCCCTGTCGCAATCACTGGCACCATGGTGGACTCCGAGCTGCAGCTCCAACTGCTGAACATCGGCACCGAGCACATTCGCATCCAGAAGGACGAGGTGTTCATGACATGCTTCCTGGAGAACATCCCTAGCGCCACCGAAGACCACGAGAGATGGCACACATCCCCAGACATCCTGGTCCGCCAGTTCCACCTGCCCAAGCGCATCGCCAAGGAGATCGTCGCCCGCTGCCAGGAGTGCAAGAGAACCACAACCTCCCCAGTGCGCGGCACCAACCCTAGAGGACGCTTCCTGTGGGAGATGGACAACACACACTGGAACAAAACCATCATTTGGGTCGCAGTGGAGACTAACAGCGGACTGGTGGAGGCTCAGGTGATTCCCGAAGAGACCGCACTGCAAGTGGCCCTGTGTATCCTCCAGCTGATCCAACGCTACACCGTCCTGCACCTGCACAGCGACAACGGACCCTGCTTCACAGCTCACCGCATCGAGAACCTGTGCAAGTACCTGGGGATCACCAAGACAACCGGCATTCCCTACAATCCTCAGAGCCAAGGAGTCGTGGAAAGAGCCCATCGCGACCTGAAGGACAGACTGGCTGCCTATCAAGGCGACTGCGAGACCGTGGAAGCTGCACTGAGCCTCGCCCTGGTCAGCCTGAACAAGAAGAGAGGAGGCATCGGCGGACACACACCCTACGAGATCTATCTGGAGAGCGAGCACACCAAGTATCAGGACCAACTGGAGCAGCAATTCAGCAAGCAGAAGATCGAGAAATGGTGCTACGTCCGCAACAGACGCAAGGAGTGGAAGGGCCCTTACAAGGTGCTGTGGGATGGCGACGGAGCTGCAGTGATCGAGGAAGAGGGCAAGACCGCTCTGTATCGCCACCGGCACATGCGCTTCATCCCACCTCCCGACAGCGATATCCAGGACGGCTCCAGCTGA

TABLE 5 Packaging Construct Transduction Efficiency Mean GFP IntensityMock  0% 0 pCligpSyn 91% 1000 pCligpSynSer 92% 1050Comparison of BIV vector mediated GFP expression in HeLa cells. BIVvectors encoding GFP was generated either by the packaging construct,pCligpSyn or by the packaging construct, pCligpSynSer were compared fortheir transduction efficiencies of HeLa cells and intensity of GFPexpression. Transduction efficiency was measured by the percentage ofthe positive HeLa cells. Mean# GFP intensity was scored by relative fluorescence intensity. Bothtransduction efficiency and mean GFP intensity were analyzed by flowcytometry analysis on a FACS Calibur (Becton Dickinson Biosciences).

We have proposed in this application that a mutation in the BIV proteasecoding region reduces the toxicity of the BIV protease to the cells.Specifically, a point mutation is made in the packaging constructpCligpSyn at the amino acid Thr coded by nucleotides ACT (correspondingto nucleotides from 1806 to 1808 in BIV viral genomic RNA isolate 127,Garvey et al., 1990). The said Thr will be replaced with Ser at the sameposition without any change in any other coding region of the packagingconstruct. This packaging construct with a Thr to Ser mutation wasdesignated as pCligpSynSer. pCligpSynSer was compared to pCligpSyn forthe ability to support BIV vector production and the transductionefficiency achieved by the BIV vectors. Specifically, 8×10⁶ 293T cellsin 10-CM dishes were transfected with pCligpSyn or pCligpSynSer (1 ug),pTracerARev (10 ug), pBIVminivec (15 ug), and pCMVVSV-G (4.5 ug).Forty-eight hours after transfection, vectors were harvested from thetransfected cells. HeLa cells were transduced with equal numbers ofvector particles as indicated by reverse transcriptase (RT) activity.Forty-eight hours after transduction, flow cytometry analysis wasperformed to score GFP positive HeLa cells. As indicated in Table 5, thevector generated by the packaging construct with the Thr to Sermutation, pCligpSynSer transduced HeLa cells as efficiently as thevector produced by the packaging construct pCligpSyn. The nucleotidesequence for this mutated gag/pol gene is shown in Table 6 (SEQ IDNO:17). TABLE 6 Sequence of recoded gag/pol with protease mutation (SeqID:17) ATGAAGCGGAGAGAGCTGGAGAAGAAACTGAGGAAAGTGCGCGTGACACCTCAACAGGACAAGTACTATACCATCGGCAACCTGCAGTGGGCCATCCGCATGATCAACCTGATGGGCATCAAGTGCGTGTGCGACGAGGAATGCAGCGCCGCTGAGGTCGCCCTGATCATCACCCAGTTTAGCGCCCTCGACCTGGAGAACTCCCCTATCCGCGGCAAGGAAGAGGTGGCCATCAAGAATACCCTGAAGGTGTTTTGGAGCCTGCTGGCCGGATACAAGCCTGAGAGCACCGAGACCGCCCTGGGATACTGGGAAGCCTTCACCTACAGAGAGAGGGAAGCTAGAGCCGACAAGGAGGGAGAGATCAAGAGCATCTACCCTAGCCTGACCCAGAACACCCAGAACAAGAAACAGACCAGCAATCAGACAAACACCCAGAGCCTGCCCGCTATCACCACACAGGATGGCACCCCTCGCTTCGACCCCGACCTGATGAAGCAGCTGAAGATCTGGTCCGATGCCACAGAGCGCAATGGAGTGGACCTGCATGCCGTGAACATCCTGGGAGTGATCACAGCCAACCTGGTGCAAGAAGAGATCAAGCTCCTGCTGAATAGCACACCCAAGTGGCGCCTGGACGTGCAGCTGATCGAGAGCAAAGTGAGAGAGAAGGAGAACGCCCACCGCACCTGGAAGCAGCATCACCCTGAGGCTCCCAAGACAGACGAGATCATTGGAAAGGGACTGAGCTCCGCCGAGCAGGCTACCCTGATCAGCGTGGAGTGCAGAGAGACCTTCCGCCAGTGGGTGCTGCAGGCTGCCATGGAGGTCGCCCAGGCTAAGCACGCCACACCCGGACCTATCAACATCCATCAAGGCCCTAAGGAACCCTACACCGACTTCATCAACCGCCTGGTGGCTGCCCTGGAAGGAATGGCCGCTCCCGAGACCACAAAGGAGTACCTCCTGCAGCACCTGAGCATCGACCACGCCAACGAGGACTGTCAGTCCATCCTGCGCCCTCTGGGACCCAACACACCTATGGAGAAGAAACTGGAGGCCTGTCGCGTGGTGGGAAGCCAGAAGAGCAAGATGCAGTTCCTGGTGGCCGCTATGAAGGAAATGGGGATCCAGTCTCCTATTCCAGCCGTGCTGCCTCACACACCCGAAGCCTACGCCTCCCAAACCTCAGGGCCCGAGGATGGTAGGAGATGTTACGGATGTGGGAAGACAGGACATTTGAAGAGGAATTGTAAACAGCAAAAATGCTACCATTGTGGCAAACCTGGCCACCAAGCAAGAAACTGCAGGTCAAAAAACGGGAAGTGCTCCTCTGCCCCTTATGGGCAGAGGAGCCAACCACAGAACAATTTTCACCAGAGCAACATGAGTTCTGTGACCCCATCTGCACCCCCTCTTATATTAGATTAGACAAACAGCCTTTTATAAAGGTGTTCATTGGCGGCCGCTGGGTGAAGGGACTGGTGGACTCAGGCGCTGACGAGGTGGTGCTGAAGAACATCCACTGGGACCGCATCAAAGGCTACCCTGGAACACCCATCAAGCAGATCGGCGTGAACGGCGTGAACGTGGCTAAGCGCAAAACACATGTGGAGTGGAGATTCAAAGACAAGACCGGCATCATTGACGTCCTCTTCAGCGACACACCTGTGAACCTGTTTGGCAGAAGCCTGCTCAGATCCATCGTGACCTGCTTTACCCTGCTGGTGCACACCGAGAAGATCGAGCCACTGCCTGTGAAGGTGCGCGGCCCTGGACCTAAGGTGCCACAATGGCCCCTGACCAAGGAGAAATACCAGGCCCTGAAGGAGATCGTGAAGGACCTGCTGGCCGAGGGAAAGATCAGCGAAGCTGCCTGGGACAACCCTTACAACACACCCGTGTTCGTGATCAAGAAGAAAGGCACCGGCCGCTGGCGCATGCTGATGGACTTCCGCGAGCTGAATAAGATCACCGTGAAAGGCCAAGAGTTCAGCACAGGACTCCCTTATCCACCCGGCATCAAGGAGTGTGAGCACCTGACCGCCATCGACATCAAGGACGCCTACTTCACCATCCCTCTGCACGAGGACTTCAGACCCTTCACAGCCTTCAGCGTGGTCCCAGTGAACCGCGAGGGCCCCATCGAGCGGTTCCAGTGGAACGTCCTGCCTCAAGGCTGGGTGTGCTCCCCTGCCATCTACCAGACCACAACCCAGAAGATCATTGAGAACATCAAGAAGAGCCATCCCGACGTGATGCTGTATCAGTACATGGATGACCTCCTGATTGGCAGCAATCGCGATGACCACAAGCAGATCGTGCAGGAGATCAGAGACAAGCTGGGCAGCTATGGCTTCAAGACACCCGACGAGAAAGTGCAGGAAGAGCGCGTGAAGTGGATCGGCTTCGAGCTGACACCTAAGAAATGGAGATTCCAGCGTAGGCAACTGAAGATCAAGAACCCACTGACCGTGAACGAACTCCAGCAGCTGGTCGGCAACTGTGTGTGGGTGCAGCCCGAGGTGAAGATCCCTCTGTACCCACTGACCGATCTGCTCCGCGACAAGACCAACCTGCAGGAAAAGATCCAGCTGACACCCGAGGCCATCAAGTGCGTGGAAGAGTTCAACCTGAAGCTGAAAGATCCCGAGTGGAAGGACAGAATTCGCGAAGGAGCCGAGCTGGTGATCAAGATCCAAATGGTCCCTCGCGGCATCGTGTTCGACCTGCTGCAAGACGGCAATCCTATCTGGGGAGGCGTGAAAGGACTGAACTACGACCACAGCAACAAGATCAAGAAGATCCTGCGCACCATGAACGAGCTGAACCGCACCGTGGTGATCATGACCGGACGCGAAGCTAGCTTTCTCCTGCCTGGATCCAGCGAGGATTGGGAGGCCGCCCTGCAGAAGGAAGAGAGCCTGACCCAAATCTTTCCCGTGAAGTTCTACCGCCATAGCTGTAGATGGACAAGCATCTGTGGACCCGTCCGCGAGAACCTGACCACCTACTATACCGACGGCGGGAAGAAAGGAAAGACAGCTGCGGCAGTGTACTGGTGTGAAGGAAGAACTAAGAGCAAAGTGTTCCCTGGAACCAATCAACAGGCTGAGCTGAAGGCAATCTGCATGGCTCTGCTGGACGGACCTCCCAAGATGAACATCATCACCGACAGCCGCTACGCTTATGAGGGCATGAGAGAGGAACCTGAGACCTGGGCTCGCGAGGGCATCTGGCTGGAGATTGCAAAGATCCTGCCATTCAAGCAATACGTCGGAGTGGGCTGGGTCCCTGCTCACAAAGGCATTGGAGGCAATACCGAGGCTGACGAAGGAGTGAAGAAAGCCCTGGAGCAAATGGCACCATGTTCCCCTCCCGAGGCTATGCTGCTCAAACCTGGCGAGAAGCAAAACCTGGAGACCGGCATCTACATGCAAGGCCTGAGACCTCAGAGCTTCCTGCCCCGCGCTGACCTCCCTGTCGCAATCACTGGCACCATGGTGGACTCCGAGCTGCAGCTCCAACTGCTGAACATCGGCACCGAGCACATTCGCATCCAGAAGGACGAGGTGTTCATGACATGCTTCCTGGAGAACATCCCTAGCGCCACCGAAGACCACGAGAGATGGCACACATCCCCAGACATCCTGGTCCGCCAGTTCCACCTGCCCAAGCGCATCGCCAAGGAGATCGTCGCCCGCTGCCAGGAGTGCAAGAGAACCACAACCTCCCCAGTGCGCGGCACCAACCCTAGAGGACGCTTCCTGTGGCAGATGGACAACACACACTGGAACAAAACCATCATTTGGGTCGCAGTGGAGACTAACAGCGGACTGGTGGAGGCTCAGGTGATTCCCGAAGAGACCGCACTGCAAGTGGCCCTGTGTATCCTCCAGCTGATCCAACGCTACACCGTCCTGCACCTGCACAGCGACAACGGACCCTGCTTCACAGCTCACCGCATCGAGAACCTGTGCAAGTACCTGGGCATCACCAAGACAACCGGCATTGCCTACAATCCTCAGAGCCAAGGAGTCGTGGAAAGAGCCCATCGCGACGTGAAGGACAGACTGGCTGCCTATCAAGGCGACTGCGAGACCGTGGAAGCTGCACTGAGCCTCGCCCTGGTCAGCCTGAACAAGAAGAGAGGAGGCATCGGCGGACACACACCCTACGAGATCTATCTGGAGAGCGAGCACACCAAGTATCAGGACCAACTGGAGCAGCAATTCAGCAAGCAGAAGATCGAGAAATGGTGCTACGTCCGCAACAGACGCAAGGAGTGGAAGGGCCCTTACAAGGTGCTGTGGGATGGCGACGGAGCTGCAGTGATCGAGGAAGAGGGCAAGACCGCTCTGTATCCCCACCGGCACATGCGCTTCATCCCACCTCCCGACAGCGATATCCAGGACGGCTCCAGCTGA

TABLE 7 Sequence of Rev gene (SEQ ID NO:16)ATGGATCAGGACCTAGACCGCGCGGAACGCGGGGAAAGGGGAGGAGGATCCGAAGAACTGCTTCAGGAGGAGATCAACGAAGGGAGGCTGACAGCCAGAGAAGCTTTACAAACATGGATCAATAACGATTCTCCTAGGTATGTTAAGAAGCTGCGCCAAGGTCAGGCAGAATTACCAACATCTCCCGGCGGAGGAGGAGGACGGGGACACAGAGCCAGAAAGCTCCCCGGCGAGAGGAGACCCGGCTTCTGGAAGTCTCTACGAGAATTGGTTGAACAAAATAGGAGAAAGCAAGAACGACGCCTATCGGGTCTGGACAGAAGAATACAACAGCTTGAGGATCTTGTTCGCCACATGTCGCTGGGATCTCCTGACCCCTCAACTCCTTCAGCTTGCGTTCTTTCTGTTAACCCTGCTGCTCAAACTCCTTTGGGACATCTTCCGCCACGCTCCTATTTTAAACTTAAAAGGGTGGACTGTGGGGCAGGGTGGGACCTCAGGACAACAGCAGCCCCCGGACTTCCCATATGTGAATTGGACTGGATCCAGG GAACAAAATAA

Example 10 One Method for Generation of Producer Cell Lines for BIVBased Lentiviral Vectors

The BIV based lentiviral packaging construct pCIBIVGPmut is transfectedinto 293Ea6 cells (a cell line expressing A-MLV envelope as described inthis invention in Example 3) together with a plasmid encoding selectablemarker puromycin as described in this invention for HIV based lentiviralpackaging construct. The transfected cells are cultured in a mediumcontaining puromycin as described in this invention in Example 3. Thepuromycin resistant single cell clones are monitored for BIV Gag/Polproduction in the cell culture medium by specifically assaying for BIVReverse Transcriptase (RT) activity. The RT assay is performed with a RTassay Kit purchased from Roche (Product No: 1828657) by taking advantageof the fact that BIV RT cross-reacts with HIV RT. Single cell cloneexpressing the highest RT is monitored for its stability in BIV Gag/Polproduction. Alternatively, other mammalian cell lines instead of 293cell line is used. Alternatively, a cell line constitutively expressingother viral envelope instead of A-MLV envelope is used. A BIV basedtransfer vector is introduced into the cell clone with highest stableproduction of BIV RT through transfection with a BIV based transfervector plasmid or infection with a BIV based lentiviral vectorparticles. Supernatants resulting from the packaging cell line arecollected at various times.

Example 11 Another Method for Generation of Producer Cell Lines for BIVBased Lentiviral Vectors

To generate a producer cell line for BIV-based lentiviral vectorproduction, a construct encoding recoded BIV Gag/Pol with the proteasemutation is co-transfected with a plasmid encoding the selectable markerhygromycin into 293 cells. Hygromycin resistant clones are selected andscreened for BIV Gag/Pol expression by reverse transcriptase (RT)activity assay. Positive clones expressing BIV Gag/pol are expanded forfunctional analysis by co-transfection with BIV Rev expression construct(pTracerARev), VSV-G expression construct, and a BIV transfer vectorconstruct encoding GFP. Forty-eight hours after transfection,supernatant from the transfected cells is collected and used totransduce naïve 293 cells.

The clones producing the highest amounts of functional BIV vectors asindicated by the percentage of GFP positive cells are saved for furtheruse. To the identified functional cell clones that express BIV Gag/Pol,a second construct encoding BIV Rev with a selectable marker puromycin(pEF1aRevIRESPuro) is introduced by transfection. Puromycin resistantclones are selected. The clones are then screened for functional BIVGag/Pol and Rev expression by co-transfection with a VSV-G expressionconstruct and a BIV transfer vector construct encoding GFP. Forty-eighthours after transfection, supernatant from the transfected cells will becollected and used to transduce naïve 293 cells. The clones producingthe highest amounts of functional BIV vectors as indicated by thepercentage of GFP positive cells are saved for further use. To thefunctional cell clones that express BIV Gag/pol and BIV Rev, a thirdconstruct encoding mutant LCMV glycoprotein (Beyer et al., J. Virol.76:1488-1495) with a selectable marker neomycin (pCILCMVgpIRESNeo) isintroduced by transfection. Neomycin (G418) resistant clones areselected. The clones are then screened for functional BIV Gag/Pol, BIVRev, and mutant LCMV glycoprotein expression by transfection with a BIVtransfer vector construct encoding GFP. The clones producing the highestamounts of functional BIV vectors as indicated by the percentage of GFPpositive cells are saved for further use. The identified clonessimultaneously expressing functional BIV Gag/Pol, BIV Rev, and mutantLCMV glycoprotein serve as a packaging cell line. To generate a producercell line for a given BIV-based vector production, a BIV-based transfervector encoding a desired transgene (marker gene or therapeutic gene) isintroduced into the packaging cell line through transfection with aBIV-based transfer vector plasmid or infection with a BIV-basedlentiviral vector particle. Supernatant obtained from the packaging cellline contains the desired BIV-based lentiviral vector.

1-22. (canceled)
 23. A method for producing a recombinant lentiviralvector particle preparation comprising the steps of: transfecting apackaging cell line comprising a packaging construct comprising asequence encoding a lentiviral protease gene containing a substitutionwhere T is replaced with S in the DTGAD motif and a deletion in alentiviral packaging signal sequence with a lentiviral vector,propagating the cell line in a suitable culture medium and obtaining arecombinant lentiviral vector particles preparation from said culturemedium.
 24. The method of claim 23, wherein said sequence encoding saidprotease comprises a lentiviral pol gene.
 25. The method of claim 24,further comprising a lentiviral gag gene.
 26. The method of claim 23, 24or 25 which is obtained from an HIV genome.
 27. The method of claim 26further comprising a deletion of, or mutation in, a vif gene, a vpr geneor both.
 28. The method of claim 23 wherein said lentiviral protease isan HIV protease.
 29. The method of claim 23, 24 or 25 which is obtainedfrom a BIV genome.
 30. The method of claim 29 further comprising adeletion of, or mutation in, one or more of a vif gene, a W gene, a Ygene or a Tat gene.
 31. The method of claim 23 wherein said lentiviralprotease is a BIV protease.
 32. The method of claim 23 wherein saidlentiviral protease is an EIAV protease.
 33. The method of claim 23wherein said lentiviral protease is an SIV protease.
 34. The method ofclaim 23 wherein said lentiviral protease is an FIV protease.
 35. Themethod of claim 23 wherein expression of the lentiviral protease gene isconstitutive.
 36. The method of claim 24 or 25 wherein expression of thelentiviral pol gene is constitutive.
 37. The method of claim 25 whereinexpression of the lentiviral gag gene is constitutive.
 38. The method ofclaim 23 wherein said cell line further comprises an env gene.
 39. Themethod of claim 23 or 38, wherein said cell line further comprises a revgene.